The overall objective of this proposal is to define the role of cellular cation movements and the physical and chemical changes in the plasma membrane during the activation of the alveolar macrophage and to determine the sequence of these events. For most phagocytic cells, changes in ion permeabilities and particularly mobilization of intracellular calcium have been implicated during activation (steps leading to superoxide production or chemotaxis). Thus far, the activation sequence of the alveolar macrophage has been the least studied among the phagocytic cells. However, since the alveolar macrophage has a significant role as a first line of host defense in the lung, research on the macrophage merits a thorough evaluation and characterization. To conduct the various aspects of this proposal, a number of radioactive, spectrophotometric and fluorimetric techniques will be employed. These include ionic flux studies (calcium-45, sodium-22, etc.), development of techniques for the continuous assay of intracellular and extracellular calcium movement (Quin2, aequorin, arsenazo III, calcium electrodes), measurement of transmembrane electrical gradients (lipophilic probes and cyanine dyes) and measurement of pH gradients. We will correlate these measurements to the physical state of the membrane (anisotropy measurements). Finally, we will try to develop a model for the relationship between membrane calcium (and cytosolic free calcium with possible regulation by calmodulin), membrane fluidity, pH gradients, transmembrane electrical gradients and their relationships to stimulated superoxide production. If the regulation of superoxide production is at the membrane level, then an understanding of this phenomenon would facilitate possible regulation of macrophage activation (e.g., superoxide production). When possible, extrapolation will be made to the human system.